Brain tumors are an important cause of cancer-related death. Malignant brain tumors are highly angiogenic and their microvessels express the integrin cell surface receptors alphavbeta3 and alphavbeta5. Clinical trials using RGDfV, an integrin-alphavbeta3/beta5 inhibitor, are promising. It is therefore critical to better understand the molecular mechanism of alphav-integrin inhibition in order to improve its therapeutic efficacy in future clinical trials. Our goal in this proposal is to examine ceramide as a molecular target in inhibition of endothelial integrins alphavbeta3/beta5. Inhibition of integrins alphavbeta3/beta5 induces apoptosis of endothelial cells (EC) and prevents brain tumor growth in an intracranial mouse xenograft model. Our previous work shows that inhibition of EC alphavbeta3/beta5 increases levels of the intracellular pro-apoptotic lipid second messenger ceramide. Our more recent results suggest this is by hydrolysis of sphingomyelin due to activation of acid sphingomyelinase. Importantly, integrins alphavbeta3/beta5 and p53 act in concert in neovascularization, but the mechanism by which integrins regulate p53 is not known. Our preliminary data, that inhibition of EC integrins alphavbeta3/beta5 suppressed phosphorylation of the pro-survival kinase PKB/Akt, and that Akt regulated p53 activity, are very interesting in this regard, especially since Akt is a potential target for inhibition by ceramide. And lastly, we have shown that the synthetic retinoid, fenretinide, also induces ceramide-mediated EC apoptosis. However, this ceramide generation is by de novo synthesis, a mechanism that is different from RGDfV. Based on these data we hypothesize that ceramide is critical in the mechanism of endothelial cell apoptosis that is induced by inhibition of integrins alphavbeta3/beta5. Important questions stemming from this are: 1) Is RGDfV-induced ceramide required for integrin alphavbeta3/beta3-mediated apoptosis; 2) Do integrins alphavbeta3/beta5 control p53 activity via their regulatory effect on Akt, and does ceramide mediate this function; 3) Does combination of RGDfV + fenretinide have better therapeutic efficacy against brain tumors, compared to each alone; and last, 4) Does the in vivo effect of RGDfV target the endothelial cells, the tumor cells, or both. Our Specific Aims to investigate these questions and examine our hypothesis are: 1.To determine molecular interactions between ceramide, integrins alphavbeta3/alphavbeta5 and endothelial cell apoptosis. 2. To elucidate the signaling mechanism of alphavbeta3/alphavbeta5 integrin blockade downstream of ceramide generation. 3.To examine the combination of fenretinide + RGDfV in an intracranial mouse brain tumor model. These studies will provide critical mechanistic data on the function of alphavbeta3/beta5 integrin inhibition in antiangiogenic cancer therapy, and will be essential for design of improved treatments for patients with malignant brain tumors.